sample-preparation-for-mass-spectrometry-based-identification-rna

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

University of Pennsylvania Perelman School of Medicine

Interactions between noncoding RNAs and chromatin proteins play important roles in gene regulation, but the molecular details of most of these interactions are unknown. Using protein-RNA photocrosslinking and mass spectrometry on embryonic stem cell nuclei, we identified and mapped, at peptide resolution, the RNA-binding regions in ∼800 known and previously unknown RNA-binding proteins, many of which are transcriptional regulators and chromatin modifiers. In addition to known RNA-binding motifs, we detected several protein domains previously unknown to function in RNA recognition, as well as non-annotated and/or disordered regions, suggesting that many functional protein-RNA contacts remain unexplored. We identified RNA-binding regions in several chromatin regulators, including TET2, and validated their ability to bind RNA. Thus, proteomic identification of RNA-binding regions (RBR-ID) is a powerful tool to map protein-RNA interactions and will allow rational design of mutants to dissect their function at a mechanistic level.

Robert Warneford-Thomson

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